This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Malaria is a devastating illness that each year infects over 500 million people, resulting in the death of over 1 million people the majority of which are pregnant women and children (WHO, 2008). There are currently no vaccines against Plasmodium falciparum, the parasite responsible for the mortality associated with this disease and drug resistance to current therapies is rapidly spreading (Chico et al., 2008). In Lazarus et al., 2008, we proposed a new model for the transport of hemoglobin from the host erythrocyte cytosol to the digestive vacuole (DV) in intracellular malaria parasites (Fig. 1). This appears to involve a double-membrane, tubular structure formed from the vacuolar membrane that surrounds the intra-erythrocytic parasite (PVM) and the parasite plasma membrane (PPM). This structure is referred to as a cytostome. We have collaborated with the RVBC at the Wadsworth Center to produce a tomogram of an infected erythrocyte that supports a key feature of our new model (Fig. 2). We have many examples from thin section transmission EM of steps 2 and 3, but previously had only captured one image that could represent steps 4 and 5. We suspect that there is a fusion event between the double membrane tubular cytostome and the parasite DV, which is required for hemoglobin transfer. The morphological characterization of the cytostomal pathway is vital to understanding key events in the hemoglobin transport process. We've demonstrated that the delivery of host cell hemoglobin to the DV of intra-erythrocytic P. falciparum parasites is an obligate parasite process, as inhibition of this route arrested parasite development and lead to the elimination of parasitemia from culture (Lazarus, Schneider, and Taraschi, 2008). Treatment with a small molecule inhibitor which targets the GTPase activity of dynamin, a protein believed to be associated with cytostome, caused the disruption of cytostome morphology and prevented hemoglobin transport to the DV, resulting in the arrest of parasite development. These results validated the cytostome feeding mechanism as a new target for antimalarial therapy. Experimental Plan: We suspect that the membranes of the cytostome and the DV fuse, but we've been unable to visualize this event. Likewise the electron dense collar that is observed around the cytosome membrane probably is involved in its formation. Its preservation in conventionally stained and embedded sections is poor, preventing us from learning anything useful about its subunit structure (possibly dynamin rings). The current tomographic reconstruction is promising but we need to move to cryogenic specimen preparative procedures to maximize the information obtained from 3D analysis. Parasitized erythrocytes could be rapidly frozen using the RVBC's BalTec high-pressure freezer, then either freeze-substituted and conventionally sectioned, or kept in the frozen hydrated state and thinned for tomographic data collection by FIB-milling (TRD1). Comparative results from the RVBC on conventionally prepared and frozen-hydrated tissue and isolated organelles give us confidence that our ability to detect evidence of membrane fusion events and substructures involved in membrane remodeling or interactions will be greatly improved with frozen-hydrated specimens (Hsieh et al., 2006;Mannella et al., 2006a;Renken et al., 2009). Full 360[unreadable] tilt range tomography (TRD1) would be a major advantage for determining the structure of the cylindrical cytosomal collar in sections cut from the freeze-substituted material. Likewise, use of phase plates to enhance contrast (TRD2) should yield better resolution with the beam-sensitive frozen-hydrated specimens than is currently possible with extreme defocus phase contrast. If the collars are identical or fall into definable classes, it will be possible to apply averaging techniques (Renken et al., 2009) which would greatly improve the resolution of the collar structure. We expect that the unique facilities and expertise available at the Wadsworth Center will help us generate 3D images of the intracellular parasite that will greatly advance our knowledge of the critical hemoglobin transport pathway. If we are successful, this information will be of great benefit to our program to identify small molecules that inhibit this process and could help guide the development of new antimalarials. References Chico, RM, R Pittrof, B Greenwood and D Chandramohan. 2008. Azithromycin-chloroquine and the intermittent preventive treatment of malaria in pregnancy Malar.J 7:255. Hsieh CE, A Leith, CA Mannella, J Frank and M Marko. 2006. Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections. J.Struct. Biol., 153:1-13. Lazarus MD, TG Schneider and TF Taraschi. 2008. A new model for hemoglobin ingestion and transport by the human malaria parasite Plasmodium falciparum J Cell Sci. 121:1937-1949. Mannella CA. 2006. Structure and dynamics of mitochondrial inner membrane cristae Biochim. Biophys. Acta 1763:542-548. WHO. World Health Organization, Global Health Atlas. (2008--pamphlet), Renken C, C Hsieh, M Marko, B Rath, A Leith, T Wagenknecht, J Frank and CA Mannella, 2009. Structure of frozen-hydrated triad junctions: a case study in motif searching inside tomograms. J. Struct. Biol. 165:53-63. (cover picture) Fig. 1: Key Events in Hemoglobin Transport to the Parasite DV. 1) Cytostome nucleation [unreadable]indentation of PVM / PPM and formation of electron dense collar. 2) Cytostome elongation and formation of a hemoglobin filled cytostome. 3) Cytostomal tube elongation [unreadable]a hemoglobin-filled tube extends to the DV. 4) PVM fusion/fission [unreadable]PVM fuses and membrane fission creates a hemoglobin containing vesicle. 5) PPM fusion/fission [unreadable]PPM fuses at the parasite surface and with the DV and delivers a single-membrane hemoglobin containing vesicle to the DV. Steps 4 and 5 are thought to be very rapid events.